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Oligodendrocyte-myelin glycoprotein (OMgp)-specific binding agents are
used to reduce OMgp-mediated axon growth inhibition. Mixtures of axons
and OMgp and mixtures of Nogo receptor (NgR) and OMgp are used in
pharmaceutical screens to characterize agents as inhibiting binding of
NgR to OMgp and promoting axon regeneration.

[0002] This work was supported by Federal Grant No. 1R21NS41999-01 from
NINDS. The government may have rights in any patent issuing on this
application.

Claims

What is claimed is:

1. A method for reducing axon growth inhibition mediated by
oligodendrocyte-myelin glycoprotein (OMgp) and detecting resultant
reduced axon growth inhibition, the method comprising steps: contacting a
mixture comprising an axon and isolated OMgp with an agent and under
conditions wherein but for the presence of the agent, the axon is subject
to growth inhibition mediated by the OMgp; and detecting resultant
reduced axon growth inhibition.

2. A method according to claim 1, wherein the isolated OMgp consists
essentially of OMgp.

3. A method according to claim 1, wherein the isolated OMgp consists
essentially of OMgp, wherein the OMgp is soluble and GPI-cleaved.

4. A method according to claim 1, wherein the OMgp is recombinantly
expressed on a surface of a cell.

5. A method according to claim 1, wherein the mixture is in vitro.

6. A method according to claim 1, wherein the agent is a candidate agent
not previously characterized to bind OMgp nor reduce axon growth
inhibition mediated by OMgp and the detecting step characterizes the
candidate agent as reducing axon growth inhibition mediated by OMgp.

7. A method according to claim 1, wherein the agent is a candidate agent
not previously characterized to reduce axon growth inhibition mediated by
OMgp and the detecting step characterizes the candidate agent as reducing
axon growth inhibition mediated by OMgp.

8. A method according to claim 1, wherein the agent comprises an
OMgp-specific antibody fragment.

9. A method according to claim 1, wherein the agent is soluble Nogo
receptor (NgR) peptide sufficient to specifically bind the OMgp and
competitively inhibit binding of the OMgp to NgR

10. A method for reducing axon growth inhibition mediated by OMgp and
detecting resultant reduced axon growth inhibition, the method comprising
steps: contacting a mixture comprising an axon and OMgp with an exogenous
OMgp-specific binding agent and under conditions wherein the agent binds
the OMgp and but for the presence of the agent, the axon is subject to
growth inhibition mediated by the OMgp, and detecting resultant reduced
axon growth inhibition.

11. A method according to claim 10, wherein the mixture is in vitro.

12. A method according to claim 10, wherein the agent is a candidate agent
not previously characterized to bind OMgp nor reduce axon growth
inhibition mediated by OMgp and the detecting step characterizes the
candidate agent as reducing axon growth inhibition mediated by OMgp.

13. A method according to claim 10, wherein the agent is a candidate agent
not previously characterized to reduce axon growth inhibition mediated by
OMgp and the detecting step characterizes the candidate agent as reducing
axon growth inhibition mediated by OMgp.

14. A method according to claim 10, wherein the agent comprises an
OMgp-specific antibody fragment.

15. A method according to claim 10, wherein the agent is soluble NgR
peptide sufficient to specifically bind the OMgp and competitively
inhibit binding of the OMgp to NgR.

16. A method according to claim 10, wherein the agent is soluble NgR
peptide sufficient to specifically bind the OMgp and competitively
inhibit binding of the OMgp to NgR, wherein the peptide consists
essentially of a sequence within SEQ ID NO: 1 at least six residues in
length.

17. A method for characterizing an agent as inhibiting binding of NgR to
OMgp, the method comprising the steps of: incubating a mixture comprising
NgR, OMgp and an agent under conditions whereby but for the presence of
the agent, the NgR and OMgp exhibit a control binding; and detecting a
reduced binding of the NgR to the OMgp, indicating that the agent
inhibits binding of the NgR to the OMgp.

18. A method according to claim 17, wherein at least one of the NgR and
OMgp is soluble and GPI-cleaved.

19. A method according to claim 17, wherein one of the NgR and OMgp is
soluble and GPI-cleaved and the other is membrane-bound.

20. A method according to claim 17, wherein at least one of the NgR and
OMgp is recombinantly expressed on a surface of a cell.

Description

[0001] This application is a continuing application under 35USC120 of
USSN10/006,002 filed on Dec. 3, 2001.

FIELD OF THE INVENTION

[0003] The invention is in the field of reducing meylin-mediated
inhibition of axon regeneration.

BACKGROUND OF THE INVENTION

[0004] The inhibitory activity associated with myelin is a major obstacle
for successful axon regeneration in the adult mammalian central nervous
system (CNS).sup.1,2. In addition to myelin associated glycoprotein
(MAG).sup.3-4 and Nogo-A.sup.5-7, evidence suggests the existence of
other inhibitors in CNS myelin.sup.8. We show that a
glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein,
oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of
neurite outgrowth. Like Nogo-A, OMgp contributes significantly to the
inhibitory activity associated with CNS myelin. To elucidate the
mechanisms that mediate this inhibitory activity of OMgp, we screened an
expression library and identified the Nogo receptor (NgR).sup.9 as a high
affinity OMgp binding protein. Cleavage of NgR and other GPI-linked
proteins from the cell surface renders dorsal root ganglion axons
insensitive to OMgp. Introduction of exogenous NgR confers
OMgp-responsiveness to otherwise insensitive neurons. We conclude that
OMgp is an physiological neurite outgrowth inhibitor that acts through
and is a physiological ligand of the NgR and its associated receptor
complex. We show that Interfering with the OMgp/NgR pathway allows
lesioned axons to regenerate after injury in vivo.

SUMMARY OF THE INVENTION

[0005] The invention provides methods and compositions for reducing
OMgp-mediated axon growth inhibition. In one embodiment, the method
comprising steps (a) contacting a mixture comprising an axon and isolated
OMgp with an agent and under conditions wherein but for the presence of
the agent, the axon is subject to growth inhibition mediated by the OMgp;
and (b) detecting resultant reduced axon growth inhibition. In an
alternative embodiment, the method comprises steps: (a) contacting a
mixture comprising an axon and OMgp with an exogenous OMgp-specific
binding agent and under conditions wherein but for the presence of the
agent, the axon is subject to growth inhibition mediated by the OMgp,
whereby the agent binds the OMgp and reduces the growth inhibition; and
(b) detecting resultant reduced axon growth inhibition.

[0006] These methods may be practiced with isolated neurons in vitro, or
with neurons in situ. Suitable agents include (i) a candidate agent not
previously characterized to bind OMgp nor reduce axon growth inhibition
mediated by OMgp; (ii) a candidate agent not previously characterized to
reduce axon growth inhibition mediated by OMgp; (iii) an OMgp-specific
antibody fragment; (iv) a soluble NgR peptide sufficient to specifically
bind the OMgp and competitively inhibit binding of the OMgp to NgR; etc.
In more particular embodiments, the recited isolated OMgp consists
essentially of OMgp, particularly wherein the OMgp is soluble and
GPI-cleaved and/or the OMgp is recombinantly expressed on a surface of a
cell.

[0007] The invention also provides methods and compositions for
characterizing an agent as inhibiting binding of NgR to OMgp. In one
embodiment, this method comprising the steps (a) incubating a mixture
comprising NgR, OMgp and an agent under conditions whereby but for the
presence of the agent, the NgR and OMgp exhibit a control binding; and
(b) detecting a reduced binding of the NgR to the OMgp, indicating that
the agent inhibits binding of the NgR to the OMgp.

[0008] The method may be practiced in a variety of alternative
embodiments, such as (i) wherein at least one of the NgR and OMgp is
soluble and GPI-cleaved; (ii) wherein one of the NgR and OMgp is soluble
and GPI-cleaved and the other is membrane-bound; (iii) wherein at least
one of the NgR and OMgp is recombinantly expressed on a surface of a
cell; etc.

[0009] The invention also provides compositions and mixtures specifically
tailored for practicing the subject methods. For example, an in vitro
mixture for use in the subject binding assays comprises NgR, OMgp and an
agent, wherein at least one of the NgR and OMgp is soluble and
GPI-cleaved. Kits for practicing the disclosed methods may also comprise
printed or electronic instructions describing the applicable subject
method.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0010] The following descriptions of particular embodiments and examples
are offered by way of illustration and not by way of limitation. Unless
contraindicated or noted otherwise, in these descriptions and throughout
this specification, the terms "a" and "an" mean one or more, the term
"or" means and/or and polynucleotide sequences are understood to
encompass opposite strands as well as alternative backbones described
herein.

[0011] In one embodiment, the invention provides a method for reducing
axon growth inhibition mediated by OMgp and detecting resultant reduced
axon growth inhibition, the method comprising steps: contacting a mixture
comprising an axon and isolated OMgp with an agent and under conditions
wherein but for the presence of the agent, the axon is subject to growth
inhibition mediated by the OMgp; and detecting resultant reduced axon
growth inhibition, indicating that the agent reduces axon growth
inhibition mediated by OMgp.

[0012] The recited axons are mammalian neuron axons, preferably adult
neural axons, which may be peripheral or, preferably CNS neuron axons. As
exemplified below, the method may be applied to neural axons in vitro or
in situ.

[0013] OMgp is a natural, mammalian CNS myelin glycoprotein (see, Habib et
al. 1998a, 1998b) which functions as a ligand of the Nogo Receptor (NgR)
on CNS axons. OMgp cDNA has been cloned from several species, including
human (Genbank Accn No. NM.sub.--002544), mouse (Genbank Accn No.
NM.sub.--019409), and cow (Genbank Accn No. S45673). Note that OMgp cDNA
encodes two alternative initiating methionine residues; compare, Genbank
Accession Nos. M63623 (human) and S67043 (mouse). OMgp may be
membrane-bound through a GPI linkage or cleaved therefrom. As exemplified
herein, OMgp may be obtained on or cleaved from naturally expressing
myelin. Also as exemplified herein, OMgp may also be expressed
recombinantly in suitable recombinant expression systems, wherein
functional expression may be confirmed by the growth cone collapsing
assays described herein.

[0014] The recited isolated OMgp is provided isolated from other
components of OMgp's natural myelin mileau, which may be effected by
purification from such components or expression of the OMgp in a
non-natural system. In particular embodiments, the isolated OMgp is
accompanied by other components which provide or interfere with or alter
the axon growth inhibitory or No binding activity of the OMgp. Preferred
isolated OMgp is purified or recombinantly expressed, particularly on a
surface of a cell.

[0015] The recited agent may be characterized as an OMgp-specific binding
agent or, particularly as applied to pharmaceutical screens, an agent not
previously characterized to bind OMgp nor reduce axon growth inhibition
mediated by OMgp, wherein the agent is a candidate agent and the
detecting step characterizes the candidate agent as reducing axon growth
inhibition mediated by OMgp. Similarly, the agent may be a candidate
agent not previously characterized to reduce axon growth inhibition
mediated by OMgp, wherein the detecting step characterizes the candidate
agent as reducing axon growth inhibition mediated by OMgp.

[0016] Detailed protocols for implementing the recited steps are
exemplified below and/or otherwise known in the art as guided by the
present disclosure. The recited contacting and detecting steps are
tailored to the selected system. In vitro systems provide ready access to
the recited mixture using routine laboratory methods, whereas in vivo
systems, such as intact organisms or regions thereof, typically require
surgical or pharmacological methods. More detailed such protocols are
described below. Similarly, the detecting step is effected by evaluating
different metrics, depending on the selected system. For in vitro binding
assays, these include conventional solid-phase labeled protein binding
assays, such as ELISA-type formats, solution-phase binding assays, such
as fluorescent polarization or NMR-based assays, etc. For cell-based or
in situ assays, metrics typically involve assays of axon growth as
evaluated by linear measure, density, host mobility or other function
improvement, etc.

[0017] In another embodiment, the invention provides a method for reducing
axon growth inhibition mediated by OMgp and detecting resultant reduced
axon growth inhibition by (a) contacting a mixture comprising an axon and
OMgp with an exogenous OMgp-specific binding agent and under conditions
wherein the agent binds the OMgp and but for the presence of the agent,
the axon is subject to growth inhibition mediated by the OMgp, and (b)
detecting resultant reduced axon growth inhibition.

[0018] This protocol may similarly be practiced with in vitro or in vivo,
particularly in situ, mixtures. Note that in this embodiment, the agent
is necessarily an exogenous OMgp-specific binding agent and the recited
OMgp need not be isolated, i.e. it may be present in the context of its
native myelin. Accordingly, this aspect of the invention provides methods
for reducing axon growth inhibition mediated by OMgp in its native
mileau. By reducing axon growth inhibition, the methods assist the repair
of axons following injury or trauma, such as spinal cord injury. In
addition, the methods may be applied to alleviate dysfunction of the
nervous system due to hypertrophy of neurons or their axonal projections,
such as occurs in diabetic neuropathy.

[0019] An OMgp-specific binding agent exogenous to an axon or mixture
comprising an axon is not naturally present with the axon or mixture. The
OMgp-specific binding agents specifically bind the OMgp of the recited
mixture and thereby functionally inhibit the axon collapse and/or NgR
binding mediated by the OMgp. Of course, as OMgp-specific, the subject
agents inherently do not cross-react with (specifically bind to)
structurally distinct NgR ligands, such as NogoA. We have exemplified
suitable OMgp binding agents from diverse structures. Initial agents were
identified by selecting high affinity OMgp binders from natural NgR
peptides. These assays identified a number of OMgp-specific NgR peptides
encompassing NgR LRR (leucine rich repeat) sequences, including the
exemplified species: hNR260/308, mNR260/308 and rNR260/308. Natural
OMgp-specific NgR peptide sequences were subject to directed
combinatorial mutation and binding analysis. Resultant synthetic-sequence
OMgp-specific peptides include the exemplified species: s1NGR260/308,
s2NR260/308 and s3NR260/308. We also used a variety of OMgp peptide
immunogens to generate OMgp-specific antibodies and antibody fragments,
including the exemplified monoclonal antibodies OM-H2276 and OM-H5831 and
the exemplified fragments OMF-H7712 and OMF-H6290. OMgp-specific binding
agents are also found in compound libraries, including the exemplified
commercial fungal extract and a synthetic combinatorial
organo-pharmacophore-biased libraries. Structural characterization of the
exemplified OMgp binding agents (XR-178892, XR-397344, XR-573632,
SY-73273M, SY-32340L and SY-95734E) is effected by conventional organic
analysis.

[0022] In a particular embodiment, the binding agent is delivered locally
and its distribution is restricted. For example, a particular method of
administration involves coating, embedding or derivatizing fibers, such
as collagen fibers, protein polymers, etc. with therapeutic agents, see
also Otto et al. (1989) J Neurosci Res. 22, 83-91 and Otto and Unsicker
(1990) J Neurosc 10, 1912-1921. The amount of binding agent administered
depends on the agent, formulation, route of administration, etc. and is
generally empirically determined and variations will necessarily occur
depending on the target, the host, and the route of administration, etc.

[0023] The compositions may be advantageously used in conjunction with
other neurogenic agents, neurotrophic factors, growth factors,
anti-inflammatories, antibiotics etc.; and mixtures thereof, see e.g.
Goodman & Gilman 's The Pharmacological Basis of Therapeutics, 9.sup.th
Ed., 1996, McGraw-Hill. Exemplary such other therapeutic agents include
neuroactive agents such as in Table 1.

[0025] In particular embodiments, the OMgp binding agent is administered
in combination with a pharmaceutically acceptable excipient such as
sterile saline or other medium, gelatin, an oil, etc. to form
pharmaceutically acceptable compositions. The compositions and/or
compounds may be administered alone or in combination with any convenient
carrier, diluent, etc. and such administration may be provided in single
or multiple dosages. Useful carriers include solid, semi-solid or liquid
media including water and non-toxic organic solvents. As such the
compositions, in pharmaceutically acceptable dosage units or in bulk, may
be incorporated into a wide variety of containers, which may be
appropriately labeled with a disclosed use application. Dosage units may
be included in a variety of containers including capsules, pills, etc.

[0026] The invention also provides pharmaceutical screens for inhibitors
of OMgp-NgR binding, particularly, methods for characterizing an agent as
inhibiting binding of NgR to OMgp by: (a) incubating a mixture comprising
NgR, OMgp and an agent under conditions whereby but for the presence of
the agent, the NgR and OMgp exhibit a control binding; and (b) detecting
a reduced binding of the NgR to the OMgp, indicating that the agent
inhibits binding of the NgR to the OMgp.

[0027] NgR is a natural, mammalian neural axon protein (Fournier et al.,
2001, Nature 409, 341-46) which functions as a receptor for Nogo66 and
for OMgp. NgR cDNA has been cloned from several species, including human
(Genbank Accn No. BC011787), mouse (Genbank Accn No. NM-022982), and rat
(Genbank Accn No. AY028438). NgR may be membrane-bound through a GPI
linkage or cleaved therefrom. As exemplified herein, NgR may be obtained
on or cleaved from naturally expressing myelin. Also as exemplified
herein, NgR may also be expressed recombinantly in suitable recombinant
expression systems, wherein functional expression may be confirmed by the
growth cone collapsing assays described herein.

[0028] The screening method is amenable to a wide variety of different
protocols. For example, in particular embodiments, at least one of the
NgR and OMgp is soluble and GPI-cleaved, one of the NgR and OMgp is
soluble and GPI-cleaved and the other is membrane-bound, and at least one
of the NgR and OMgp is recombinantly expressed on a surface of a cell.

[0029] The invention also provides compositions and mixtures specifically
tailored for practicing the subject methods. For example, an in vitro
mixture for use in the subject binding assays comprises premeasured,
discrete and contained amounts of NgR, OMgp and an agent, wherein at
least one of the NgR and OMgp is soluble and GPI-cleaved. Kits for
practicing the disclosed methods may also comprises printed or electronic
instructions describing the applicable subject method.

EXAMPLES

[0030] OMgp is a Physiological Nogo Receptor Ligand and Inhibitor of
Neurite Outgrowth. To examine whether any GPI-linked proteins in CNS
myelin may act as inhibitors of neurite outgrowth, we treated purified
bovine white matter myelin with phosphatidylinositol-specific
phospholipase C (PI-PLC) and examined the released proteins for their
ability to alter growth cone morphology in a growth cone collapse assay
using embryonic day 13 chick dorsal root ganglia (E13 DRG).sup.6,9,10. We
found that PI-PLC-released CNS myelin proteins, when added to the DRG
culture medium, exhibited potent growth cone collapsing activity. To
further characterize the inhibitory activity in the PI-PLC-released
proteins, we analyzed solubilized proteins by SDS-PAGE and silver
staining and found that a band of approximately 110 kDa in size was
significantly enriched in this fraction. Since the size of this band was
similar to that of a previously identified CNS myelin protein
OMgp.sup.11,12, we used antibodies specific for OMgp to detect enrichment
of cleaved OMgp in the PI-PLC supernatants by Western blot. Anti-OMgp
antibodies detected a band of comparable size in the PI-PLC-treated
supernatants, indicating that OMgp is a component released from CNS
myelin by PI-PLC. Next, we examined whether purified recombinant OMgp
protein was able to act as an inhibitor of neurite outgrowth in two
well-established in vitro assays, the growth cone collapse.sup.6,9,13 and
the neurite outgrowth assays.sup.3-7,9. Similar to the PI-PLC-treated
myelin supernatants, purified recombinant polyhistidine-tagged OMgp
protein (OM-his), but not proteins purified from control
vector-transfected COS-7 cells, induced the collapse of growth cones
derived from E13 chick DRG neurons in a dose-dependent manner. The
effective concentration for half-maximal response (EC.sub.50) for OM-his
was approximately 1.5 nM. Consistent with it being an inhibitor of
neurite outgrowth, the GPI-anchored OMgp protein was found to be highly
expressed by myelin basic protein (MBP)-positive mature oligodendrocytes
and enriched in the axon-adjacent myelin layers.sup.11,12 Moreover,
OM-his, when presented either as an immobilized substrate or in a soluble
form, inhibited neurite outgrowth of cerebellar granule neurons (CGN)
from postnatal day 7-9 (P7-9) rats, also in a dose-dependent manner. The
OMgp-induced inhibitory responses were similar to that brought about by
treatment of the same neurons with an alkaline phosphatase (AP) fusion
protein containing the 66 amino acid extracellular domain of Nogo-A
(AP-66).sup.6,9, indicating that OMgp, like Nogo-66, is a potent neurite
outgrowth inhibitor.

[0031] To further examine the functional importance of OMgp as a CNS
myelin associated inhibitor, we used peanut agglutinin (PNA)-Agarose
beads.sup.11 to specifically deplete OMgp, but not Nogo-A or MAG, from
.beta.-octylglucoside-solublized CNS myelin. We found that while the
inhibitory activity in the PNA-depleted myelin was significantly reduced
compared to control myelin, the OMgp-enriched eluates from the same PNA
column displayed a potent inhibitory activity. We next compared the
relative contributions of OMgp with two other known inhibitors, MAG and
Nogo-A, to the inhibitory activity associated with CNS Myelin. Anion
exchange chromatography was able to biochemically separate MAG and
Nogo-A.sup.3,15, but failed to separate OMgp from Nogo-A. Thus, we
applied the OMgp-depleted PNA column flow-through myelin fractions onto a
Q-Sepharose column and generated fractions enriched in MAG or Nogo-A by
sequential elution of the column with increasing concentrations of NaCl.
Our data revealed that the fractions enriched in MAG or Nogo-A, similar
to the OMgp-enriched eluates from the PNA column, all significantly
inhibited neurite outgrowth. Comparisons of the EC.sub.50 for each of
these fractions allowed us to estimate that the OMgp-enriched fraction
inhibited neurite outgrowth to a similar extent as the Nogo-A-enriched
fraction, but much stronger than the MAG-enriched-fraction. Our result is
consistent with previous observations that the major inhibitory activity
of CNS myelin resides in the 0.30 to 0.5 M NaCl elution fractions of
Sepharose Q columns.sup.15,16, as the majority of OMgp co-fractionates
with Nogo-A in this chromatography procedure.

[0032] To investigate the mechanisms by which OMgp inhibits neurite
outgrowth, we used an expression cloning strategy.sup.9,13,16 to identify
cell surface OMgp-binding proteins. An AP fusion protein containing OMgp
(AP-OM) was able to bind to the surface of rat P9 CGNs and to induce
collapse of E13 chicken DRG growth cones, and thus was used in the cell
surface binding assays. From pools of an adult human brain cDNA
expression library, we isolated two cDNAs that encoded OMgp-binding
proteins. Sequence analysis revealed that both cDNAs contained the
full-length coding region of the Nogo-66 receptor (NgR), which had been
previously identified as a high-affinity receptor for the extracellular
domain of Nogo-A.sup.9. We then established a CHO cell line stably
expressing the NgR and determined the binding affinity of expressed NgR
for AP-OM as 5 nM, similar to what had been determined for Nogo-66 (7 nM,
ref. 9). These data indicate that NgR is a high-affinity OMgp-binding
protein. We next performed co-precipitation experiments by incubating GST
or a GST fusion protein containing the entire extracellular domain of NgR
(GST-NgR) with OM-his alone or in the presence of AP or AP-66 protein. We
found that GST-NgR, but not the control GST protein, bound to OM-his,
indicating a direct interaction between NgR and OMgp. We next determined
which domain(s) of OMgp was responsible for binding to NgR. Like NgR,
OMgp is also a GPI-linked protein containing a leucine-rich repeat (LRR)
domain. An AP fusion protein containing only the LRR domain of OMgp
(AP-LRR) was found to be sufficient to bind strongly to NgR-expressing
cells. In addition, the C-terminal domain with serine-threonine repeats
(AP-S/T) alone was also able to bind, though less strongly, to NgR
expressing cells.

[0033] To further determine how NgR interacted at the molecular level with
these two inhibitors, we made a series of deletion constructs of NgR and
found that both the LRR and the C-terminal LRR (LRRCT) domains of NgR
were required for highest binding to OMgp and that OMgp and Nogo-66
appear to bind overlapping regions of NgR. Consistently, in both cell
surface binding and the co-precipitation assay, AP-66 and OM-his proteins
competed for binding to NgR. To examine the functional consequences of
the molecular interaction of the two ligands with NgR, we compared the
collapsing activity of OM-his plus AP-66 with that of OM-his or AP-66
alone. The estimated EC.sub.50 for OM-his plus Nogo-66 (2.5 nM) was
similar to that of OM-his (1.5 nM) and AP-66 (2.3 nM), indicating an
additive effect between OM-his and AP-66 in inducing growth cone
collapse. As the binding affinities of both OMgp and Nogo-66 to NgR are
similar, our results together imply that these two myelin components act
independently through NgR to inhibit neurite outgrowth.

[0034] As the GPI-linked NgR protein can be released by PI-PLC, we next
examined whether PI-PLC treatment could affect axonal responsiveness to
OMgp. Consistent with a previous study.sup.9, PI-PLC treatment did not
alter the growth cone morphology of E13 chick DRG neurons, but rendered
these axons insensitive to Nogo-66. Similarly, PI-PLC treatment also
abolished the growth cone-collapsing activity of OMgp. As a control, the
growth cone collapsing activity of Semaphorin 3A (Sema 3A).sup.10,13,
known to be mediated by transmembrane receptor molecules including
neuropilin-1 and members of the plexin family.sup.18, was not affected by
PI-PLC treatment. Even though PI-PLC also cleaves other GPI-anchored
proteins on the axonal surface, these results indicated that GPI-anchored
proteins, such as NgR, act as necessary signal transducers of the
inhibitory activity of OMgp.

[0035] To assess whether NgR is capable of mediating OMgp-induced
inhibitory activity on neurite outgrowth, we next took a gain-of-function
approach to examine whether expression of NgR was able to confer
OMgp-responsiveness to otherwise insensitive neurons. It has been shown
previously that chick E7 retinal ganglion neurons (RGN) are insensitive
to Nogo-66, but that introduction of exogenous NgR in these neurons
rendered their growth cones to be responsive to Nogo-66.sup.9. Using the
same strategy, we made a recombinant herpes simplex virus (HSV) that
drives expression of a FLAG-tagged full-length human NgR (FLAG-NgR) in
infected neurons. Upon infection, 80% of the E7 RGNs expressed the
FLAG-NgR protein as assessed by immunocytochemistry with an anti-FLAG
antibody. No significant morphological changes were observed in the
HSV-infected neurons. Consistent with a previous study.sup.9, expression
of FLAG-NgR conferred a growth cone collapse response to Nogo-66 in E7
RGNs. Furthermore, the growth cones of NgR-expressing axons also become
collapsible by OMgp. In contrast, a control virus driving the expression
of .beta.-galactosidase did not alter the axonal responses of the same
neurons to either Nogo-66 or OMgp. Taken together, our results indicate
that, like Nogo-66, OMgp acts through NgR and its associated receptor
complex to inhibit axon outgrowth. As opposed to Nogo-A, the majority of
which is localized intracellularly.sup.5-7, OMgp is predominantly
localized on the surfaces of oligodendrocytes and axon-adjacent myelin
layers.sup.11,12,14, indicating that OMgp is a physiological ligand of
NgR.

[0036] Purification, PI-PLC Treatment, and OMgp Depletion of Myelin.
Myelin was prepared from white matter of bovine brain according to
established protocols.sup.19. In brief, white matter tissues were
homogenized in 0.32 M sucrose in phosphate-buffered saline (PBS) and the
crude myelin that banded at the interphase of a discontinuous sucrose
gradient (0.32M/0.85M) was collected and purified by two rounds of
osmotic shock with distilled water and re-isolation over the sucrose
gradient. For PI-PLC treatment, aliquots of myelin suspensions in water
(10 mg/ml) were incubated with or without 2.5 U/ml PI-PLC (Sigma) at
37.degree. C. for 2hr, prior to centrifugation (360,000 g for 60 min).
The supernatants were concentrated, partitioned in Triton X-114, and used
for assays and for detection with Western analysis.

[0037] To deplete OMgp, myelin was first solublized with 1% octylglucoside
and the resultant extract was passed twice through columns with
PNA-Agarose (Vector Laboratories) or control Agarose beads as described
previously.sup.12. The OMgp-enriched fraction was obtained by eluting the
PNA-Agarose column with buffer containing 0.5 M D-galactose. As a
significant portion of OMgp co-fractionated with Nogo-A in anion exchange
columns.sup.12, we enriched MAG or Nogo-A from myelin by applying the
flow-through fractions from the PNA-Agarose columns onto a Q-Sepharose
(Sigma) column.sup.15. The column was then eluted stepwise with equal
amount of buffers containing 0.15 M (MAG-enriched), 0.45M (Nogo-A
enriched), or 1.0 M NaCl.sup.15. Aliquots of individual fractions were
tested for their inhibitory activity in the neurite outgrowth assay as
described previously.sup.15,16,21.

[0038] Expression Cloning and Binding Experiments. Sequences encoding
mouse OMgp were amplified from Marathon-ready mouse cDNA (Clontech) and
confirmed by sequencing analysis, prior to subcloning into the expression
vector AP-5.sup.9 for expressing an AP-OM fusion protein tagged with both
a polyhistidine and a myc epitope. The resultant plasmid DNA was
transfected into COS-7 cells and the secreted protein purified using
nickel-Agarose resins (Qiagen).

[0040] For expression cloning of OMgp-binding proteins, pools of 5,000
arrayed clones from a human brain cDNA library (Origene Technologies,
Rockville, Md.) were tranfected into COS-7 cells, and AP-OM binding was
assessed as above. We isolated single NgR cDNA clones by sub-dividing the
pools and sequencing analysis.

[0041] Generation of Recombinant Proteins and Viruses and
Co-precipitation. To express recombinant OMgp for function assays, we
subcloned the coding region of mouse OMgp (amino acids 23-392) into
pSecTag B (Invitrogen) to express his-tagged OMgp protein (OM-his) in
COS-7 cells. The expressed OM-his protein was purified using a nickel
resin. To construct recombinant herpes simplex viruses (HSV), cDNAs for
FLAG-tagged NgR or b-galatosidase were inserted into the HSV amplicon
HSV-PrpUC and packaged into the virus using helper 5dl1.2, as described
previously.sup.20. The resultant viruses were purified on sucrose
gradients, pelleted, and resuspended in 10% sucrose. The titer of the
viral stocks was .about.4.0.times.10.sup.7 infectious units/ml. For each
study, aliquots from the same batches of viral vectors were used. In
order to produce recombinant Nogo-66 protein, the sequence of Nogo-66 was
amplified from a human cDNA clone, KIAA0886, from the Kazusa DNA Research
Institute and used to generate a construct expressing the AP-66 protein
as described by GrandPre et al.sup.6. Antibodies against Nogo-A and MAG
were purchased from Alpha Diagostics and R & D Systems, respectively.

[0042] In co-precipitation experiments, 2 mg GST or GST-NgR were first
immobilized to glutathione-Agarose beads and the beads were further
incubated with or without 1 mg OM-his in the presence of 2 mg of AP or
AP-66 at 4.degree. C. for 2 hr. After extensive washing, the bound
proteins were resolved with SDS-PAGE and detected by Western blotting.

[0043] Growth Cone Collapse and Neurite Outgrowth Assays. Chick E13 DRG
and E7 retina were isolated and cultured as described
previously.sup.9,10. DRG explants cultured overnight were used for growth
cone collapse assays. To assess the effects of PI-PLC treatment,.
cultures were pre-incubated with 2 U/ml PI-PLC for 30 min prior to
treatment with individual test proteins for an additional 30 min. To
express NgR in E7 retinal ganglion neurons, we infected the explants with
recombinant HSV for 24 hr. After incubation with each test protein for 30
min, retinal explants were fixed in 4% paraformaldehyde and 15% sucrose.
Infection of HSV-LacZ was detected by a standard b-galatosidase staining
protocol.sup.20. FLAG-NgR expression was detected by incubating
paraformaldehyde-fixed cultures with M2 anti-FLAG antibody (Sigma). Bound
antibody was detected by incubation with AP-conjugated anti-rabbit IgG
second antibody and reaction with NBT and BCIP (Vector labs). Growth cone
collapse was quantified only in those positively stained for
b-galatosidase or immunoreactive for the FLAG epitope.

[0044] Neurite outgrowth assays were performed as described
previously.sup.15,21. Briefly, P7-9 rat CGNs were dissected and then
plated at a density of 1.times.10.sup.5 cells per well. The cells were
cultured for 24 hr prior to fixation with 4% paraformaldehyde and
staining with a neuronal specific anti-b-tubulin antibody (TuJ-1,
Covance). Quantification of neurite length and statistical analysis were
performed as described previously.sup.22.

[0045] Oligodendrocyte precursor cells were isolated from the cerebral
hemispheres of P1 rats and differentiated in vitro as described.sup.23.
Immunostaining of mature oligodendrocytes was performed using antibodies
against MBP (Sigma) and OMgp.sup.14.

[0046] Exemplary OMgp Binding (OMgp-NgR Binding Inhibitory) Agents. An
AP-OMgp fusion protein, prepared as described above, was used to evaluate
the OMgp binding affinity of a variety of candidate binding agents as
measured by the ability of agents preincubated with OMgp to inhibit
subsequent OMgp-NgR binding. The selected binding assay formats are
guided by structural requirements of the candidate agents and include
COS-expression, solid phase ELISA-type assay, and fluorescent
polarization assays. Candidate agents were selected from natural and
synthetic peptide libraries biased to natural NgR LRR (supra) sequences,
OMgp-specific monoclonal antibody (Mab) and Mab fragment libraries, a
commercial fungal extract library, and a synthetic combinatorial
organo-pharmacophore-biased library. In each instance, we assay specific
binding inferentially by evaluating the affect of preincubating the OMgp
with the agent, on subsequent OMgp-NgR binding. Selected exemplary high
affinity OMgp-specific binding agents subject to in vivo activity assays
(below) are shown in Table 2.

[0047] Corticospinal Tract (CST) Regeneration Assay. High affinity OMgp
binding agents demonstrating inhibition of OMgp-mediated in vitro axon
growth cone collapse as described above are assayed for their ability to
improve corticospinal tract (CST) regeneration following thoracic spinal
cord injury by promoting CST regeneration into human Schwann cell grafts
in the methods of Guest et al. (1997, supra). For these data, the human
grafts are placed to span a midthoracic spinal cord transection in the
adult nude rat, a xenograft tolerant strain. OMgp binding agents
determined to be effective in in vitro collapse assays are incorporated
into a fibrin glue and placed in the same region. Anterograde tracing
from the motor cortex using the dextran amine tracers, Fluororuby (FR)
and biotinylated dextran amine (BDA), are performed. Thirty-five days
after grafting, the CST response is evaluated qualitatively by looking
for regenerated CST fibers in or beyond grafts and quantitatively by
constructing camera lucida composites to determine the sprouting index
(SI), the position of the maximum termination density (MTD) rostral to
the GFAP-defined host/graft interface, and the longitudinal spread (LS)
of bulbous end terminals. The latter two measures provide information
about axonal die-back. In control animals (graft only), the CST do not
enter the SC graft and undergo axonal die-back. As shown in Table 3, the
exemplified binding agents dramatically reduce axonal die-back and cause
sprouting and these in vivo data are consistent with the corresponding
growth cone collapsing activity.

[0049] The sciatic nerves of rats are sharply transected at mid-thigh and
guide tubes containing the test substances with and without guiding
filaments sutured over distances of approximately 2 mm to the end of the
nerves. In each experiment, the other end of the guide tube is left open.
This model simulates a severe nerve injury in which no contact with the
distal end of the nerve is present. After four weeks, the distance of
regeneration of axons within the guide tube is tested in the surviving
animals using a functional pinch test. In this test, the guide tube is
pinched with fine forceps to mechanically stimulate sensory axons.
Testing is initiated at the distal end of the guide tube and advanced
proximally until muscular contractions are noted in the lightly
anesthetized animal. The distance from the proximal nerve transection
point is the parameter measured. For histological analysis, the guide
tube containing the regenerated nerve is preserved with a fixative. Cross
sections are prepared at a point approximately 7 mm from the transection
site. The diameter of the regenerated nerve and the number of myelinated
axons observable at this point are used as parameters for comparison.

[0050] Measurements of the distance of nerve regeneration document
therapeutic efficacy. Similarly, plots of the diameter of the regenerated
nerve measured at a distance of 7 mm into the guide tube as a function of
the presence or absence of one or more binding agents demonstrate a
similar therapeutic effect of all 16 tested. No detectable nerve growth
is measured at the point sampled in the guide tube with the
matrix-forming material alone. The presence of guiding filaments plus the
matrix-forming material (no agents) induces only very minimal
regeneration at the 7 mm measurement point, whereas dramatic results, as
assessed by the diameter of the regenerating nerve, are produced by the
device which consisted of the guide tube, guiding filaments and binding
agent compositions. Finally, treatments using guide tubes comprising
either a matrix-forming material alone, or a matrix-forming material in
the presence of guiding filaments, result in no measured growth of
myelinated axons. In contrast, treatments using a device comprising guide
tubes, guiding filaments, and matrix containing binding agents
compositions consistently result in axon regeneration, with the measured
number of axons being increased markedly by the presence of guiding
filaments.

[0051] OMgp-Specific Monoclonal Antibodies Promote Axon Regeneration In
Vivo. In these experiments, our OM-H2276 and OM-H5831 OMgp-specific
monoclonal antibodies are shown to promote axonal regeneration in the rat
spinal cord. Tumors producing our OMgp-specific antibodies, implantation
protocols and experimental design are substantially as used for IN-1 as
described in Schnell et al., Nature 1990 January 18;343(6255):269-72. In
brief, our OM-H2276 and OM-H5831 monoclonal antibodies are applied
intracerebrally to young rats by implanting antibody-producing tumours.
In 2-6-week-old rats we make complete transections of the corticospinal
tract, a major fibre tract of the spinal cord, the axons of which
originate in the motor and sensory neocortex. Previous studies have shown
a complete absence of cortico-spinal tract regeneration after the first
postnatal week in rats, and in adult hamsters and cats. In our treated
rats, significant sprouting occurs at the lesion site, and fine axons and
fascicles can be observed up to 7-11 mm caudal to the lesion within 2-3
weeks. In control rats, a similar sprouting reaction occurs, but the
maximal distance of elongation rarely exceeded 1 mm. These results
demonstrate the capacity for CNS axons to regenerate and elongate within
differentiated CNS tissue after neutralization of OMgp-mediated axon
growth inhibition.

[0054] Fab fragment or antibody injections are performed as previously
described (Zagrebelsky et al., 1998). Animals are placed in a stereotaxic
apparatus, and the dorsal cerebellar vermis exposed by drilling a small
hole on the posterosuperior aspect of the occipital bone. The meninges
are left intact except for the small hole produced by the injection
pipette penetration. In test rats a recombinant Fab fragment of the
OM-H2276 and OM-H583 1antibodies (produced in E. coli), which neutralizes
OMgp-associated axon growth cone collapse in vitro is injected into the
cerebellar parenchyma. Three 1 .mu.l injections of Fab fragments in
saline solution (5 mg/ml) are performed 0.5-1 mm deep along the
cerebellar midline into the dorsal vermis (lobules V-VII). The injections
are made by means of a glass micropipette connected to a PV800 Pneumatic
Picopump (WPI, New Haven, Conn.). The frequency and duration of pressure
pulses are adjusted to inject 1 .mu.l of the solution during a period of
.about.10 mim. The pipette is left in situ for 5 additional minutes to
avoid an excessive leakage of the injected solution. As a control, an
affinity-purified F(ab').sub.2 fragment of a mouse anti-human IgG
(Jackson ImmunoResearch Laboratories, West Grove, Pa.) is applied to
another set of control rats using the same procedure. Survival times for
these two experimental sets are 2, 5, 7 and 30 d (four animals for each
time point). An additional set of intact animals are examined as
untreated controls.

[0055] Histological procedures. At different survival times after surgery,
under deep general anesthesia (as above), the rats are transcardially
perfused with 1 ml of 4% paraformaldehyde in 0.12 M phosphate buffer, pH
7.2-7.4. The brains are immediately dissected, stored overnight in the
same fixative at 4.degree. C., and finally transferred in 30% sucrose in
0.12 M phosphate buffer at 4.degree. C. until they sink. The cerebella
are cut using a freezing microtome in several series of 30-.mu.m-thick
sagittal sections. One series is processed for NADPH diaphorase
histochemistry. These sections are incubated for 3-4 hr in darkness at
37.degree. C. in a solution composed of -NADPH (1 mg/ml, Sigma, St.
Louis, Mo.) and nitroblue tetrazolium (0.2 mg/ml, Sigma) in 0.12 M
phosphate buffer with 0.25% Triton X-100. In some cases (two animals per
treated and control sets at 2 and 5 d survival), microglia are stained by
incubating one section series with biotinylated Griffonia simplicifolia
isolectin B4 [1:100 in phosphate buffer with 0.25% Triton X-100; Sigma
(Rossi et al., 1994a)] overnight at 4.degree. C. Sections are
subsequently incubated for 30 min in the avidin-biotin-peroxidase complex
(Vectastain, ABC Elite kit, Vector, Burlingame, Calif.) and revealed
using the 3,3' diaminobenzidine (0.03% in Tris HCl) as achromogen.

[0056] All of the other series are first incubated in 0.3% H.sub.2O.sub.2
in PBS to quench endogenous peroxidase. Then, they are incubated for 30
min at room temperature and overnight at 4.degree. C. with different
primary antibodies: anti-calbindin D-28K (monoclonal, 1:5000, Swant,
Bellinzona, Switzerland), to visualize Purkinje cells; anti-c-Jun
(polyclonal, 1:1000, Santa Cruz Biotechnology, Santa Cruz, Calif.); and
anti-CD11b/c (monoclonal OX-42, 1:2000, Cedarlane Laboratories, Homby,
Ontario) to stain microglia. All of the antibodies are diluted in PBS
with 0.25% Triton X-100 added with either normal horse serum or normal
goat serum depending on the species of the second antibody.
Immunohistochemical staining is performed according to the
avidin-biotin-peroxidase method (Vectastain, ABC Elite kit, Vector) and
revealed using the 3,3' diaminobenzidine (0.03% in Tris HCl) as a
chromogen. The reacted sections are mounted on chrome-alum gelatinized
slides, air-dried, dehydrated, and coverslipped.

[0057] Quantitative analysis. Quantification of reactive Purkinje cells in
the different experiments is made by estimating the neurons labeled by
c-Jun antibodies as previously described (Zagrebelsky et al., 1998). For
each animal, three immunolabeled sections are chosen. Only vermal
sections close to the cerebellar midline that contain the injection sites
are considered. The outline of the selected sections is reproduced using
the Neurolucida software (MicroBrightField, Colchester, Vt.) connected to
an E-800 Nikonmicroscope, and the position of every single-labeled cell
carefully marked. The number of labeled cells present in the three
reproduced sections is averaged to calculate values for every individual
animal, which are used for statistical analysis carried out by Student's
test.

[0058] A morphometric analysis of Purkinje axons in the different
experimental conditions for each animal, is performed using three
anti-calbindin-immunolabeled sections, contiguous to those examined for
c-Jun, as described in Buffo et al. (supra). Morphometric measurements
are made on 200.times.250 .mu.m areas of the granular layer chosen by
superimposing a grid of this size on the section. The selected areas
encompass most of the granular layer depth and contain only minimal
portions of Purkinje cell layer or axial white matter. In each of the
selected sections is sampled one area from the dorsal cortical lobules
and one from the ventral cortical lobules. In addition, to sample from
the different parts of these two cortical regions, areas from different
lobules are selected in the three sections belonging to each individual
animal, one area in each of lobules V, VI, and VII and one in lobules I,
II, and IX. All of the anti-calbindin-immunolabeled Purkinje axon
segments contained within the selected areas are reproduced using the
Neurolucida software (MicroBrightField) connected to an E-800 Nikon
microscope with 20.times. objective, corresponding to 750.times.
magnification on the computer screen. Each labeled axon segment or branch
is reproduced as a single profile. From these reproductions the software
calculates the number of axon profiles, their individual length, and the
total length of all the reproduced segments, the mean profile length
(total length/number of profiles), and the number of times that the axons
crossed a 25.times.25 .mu.m grid superimposed on the selected area. Data
calculated from the different areas in the three sections sampled from
each cerebellum are averaged to obtain values for every individual
animal. Statistical analysis is performed on the latter values (n=4 for
all groups at all time points) by Student's t test and paired t test.

[0084] The foregoing descriptions of particular embodiments and examples
are offered by way of illustration and not by way of limitation. All
publications and patent applications cited in this specification and all
references cited therein are herein incorporated by reference as if each
individual publication or patent application or reference were
specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in the art in light
of the teachings of this invention that certain changes and modifications
may be made thereto without departing from the spirit or scope of the
appended claims.